HK1118713B - Diagnostic compounds - Google Patents

Description

Radiofluorinated peptides

The present invention relates to novel peptide-based compounds and their use for diagnostic imaging using Positron Emission Tomography (PET). More specifically, the invention relates to the use of such peptide-based compounds as targeting vectors that bind to receptors associated with angiogenesis, in particular integrin receptors, such as the α v β 3 integrin receptor. Such compounds may therefore be used in the diagnosis or treatment of, for example, malignant diseases, heart diseases, endometriosis, inflammation-related diseases, rheumatoid arthritis and Kaposi's sarcoma. In addition, it relates to methods and reagents for preparing such peptide-based compounds.

The present radiolabeled bioactive peptides for diagnostic imaging have gained importance in nuclear medicine. Biologically active molecules that selectively interact with specific cell types can be used to deliver radioactivity to target tissues. For example, radiolabeled peptides have significant potential for the release of radionuclides to tumors, infarcts, and infected tissues for diagnostic imaging and radiotherapy.¹⁸F has a half-life of about 110 minutes and is positive for many receptor imaging studiesSelection of sub-radionuclides. Thus, because of their use in PET quantification and characterization of various diseases,¹⁸f-labeled bioactive peptides have great clinical potential.

New blood vessels can be formed by two different mechanisms: angiogenesis (vasculogenesis) or angiogenesis (angiogenesis). Angiogenesis is the formation of new blood vessels from existing blood vessel branches. The primary stimulus for this process may be inadequate nutrient and oxygen (oxygen deficiency) supply to the cells in the tissue. Cells can respond by secreting various angiogenic factors; an example which is often mentioned is Vascular Endothelial Growth Factor (VEGF). These factors cause secretion of proteolytic enzymes that cleave basement membrane proteins, and inhibitors that limit the action of these potentially harmful enzymes. Other prominent effects of angiogenic factors are to cause endothelial cell migration and division. Endothelial cells attached to the basement membrane, which form a continuous sheet on the conplus side around the blood vessel, do not undergo mitosis. The combined effect of adhesion loss and signaling from angiogenic factor receptors causes endothelial cells to move, multiply and rearrange themselves, eventually synthesizing a basement membrane around the new blood vessels.

Angiogenesis is prominent in tissue growth and remodeling, including wound healing and inflammatory processes. In order to maintain the growth rate of tumors as they reach millimeter size, angiogenesis must begin. Angiogenesis is accompanied by characteristic changes in endothelial cells and their environment. In addition to the various proteins involved in influencing and controlling proteolysis, the surface of these cells is remodeled in preparation for movement, and the cryptic structures are exposed where the basement membrane is degraded. In the case of tumors, the resulting vascular network is often disrupted, forming significant kinks and also arteriovenous shunts. Inhibition of angiogenesis is also considered to be a promising strategy for anti-tumor therapy. The transformation associated with angiogenesis is also very promising for diagnosis, an obvious example being malignant disease, but this concept also points out very promising for inflammation and various inflammation-related diseases, including atherosclerosis, macrophages of early atherosclerotic lesions may be a potential source of angiogenic factors.

Many ligands involved in cell adhesion contain the tripeptide sequence arginine-glycine-aspartic acid (RGD). The RGD sequence appears to act as a primary recognition site between the ligand representing the sequence and the receptor on the cell surface. It is generally believed that secondary interactions between ligands and receptors enhance the specificity of the interaction. These secondary effects may occur in the portion between the ligand and the receptor immediately adjacent to the RGD sequence or at sites distant from the RGD sequence.

Efficient targeting and imaging of integrin receptors associated with angiogenesis in vivo therefore requires selective, high affinity RGD-based vectors that are chemically robust and stable. Furthermore, the route of excretion is an important factor when designing imaging agents to reduce problems associated with the background.

WO 03/006491 describes peptide-based compounds targeting integrin receptors associated with angiogenesis. However, there is a need for additional peptide-based compounds that can be used in diagnostic imaging techniques such as PET. Co-pending International application PCT/GB2004/001052 describes a marker having a sequence of nucleotides suitable for use in a marker¹⁸A biologically active carrier of F. There is also a need for peptide-based compounds that can be prepared quickly and efficiently, and that also possess desirable biological activity.

In a first aspect, the present invention provides a method of radiofluorination comprising reaction of a compound of formula (I):

wherein the vector comprises the fragment:

and a compound of formula (II):

wherein:

n is an integer of 0 to 20;

m is an integer of 0 to 10;

y is H, C_1-6Alkyl (e.g. methyl), or phenyl

To obtain a compound of formula (III):

wherein m, n and Y are as defined for the compound of formula (II) and the carrier is as defined for the compound of formula (I).

This reaction may be carried out in a suitable solvent, for example in an aqueous buffer at a pH in the range 1 to 11, suitably 2 to 11, more suitably 2 to 6, and at a non-terminal temperature in the range 5 to 100 ℃, suitably 20 to 70 ℃, preferably at ambient temperature.

In a particular aspect, the carrier in formula (I) or (III) is formula (a):

wherein X⁷is-NH₂Or

Wherein a is an integer of 1 to 10, preferably a is 1.

The linker forming the carrier moiety in the compound of formula (I) is selected to provide good in vivo pharmacokinetics, as is the advantageous excretory properties of the final conjugate of formula (III). The use of linker groups with different lipophilicities and or charges can significantly alter the pharmacokinetics of the peptide in vivo to suit the needs of the diagnosis. For example, a hydrophilic linker is used when clearance of the conjugate of formula (III) from the body via renal excretion is desired, and a hydrophobic linker is used when clearance via hepatobiliary excretion is desired. The linker includes a polyethylene glycol moiety, which has been found to be promising in some cases for slowing blood clearance.

The linker-forming moiety of the carrier in the compound of formula (I) is C_1-60A hydrocarbyl radical, suitably C_1-30The hydrocarbyl group, optionally includes 1 to 30 heteroatoms, suitably 1 to 10 heteroatoms such as oxygen or nitrogen. Suitable linking group groups include alkyl, alkenyl, alkynyl chains, aromatic, polyaromatic and heteroaromatic rings, and the polymer comprises ethylene glycol, amino acid or carbohydrate subunits. Preferably the linker-forming moiety of the carrier in the compound of formula (I) comprises a polyethylene glycol subunit, and most preferably the linker is of formula B:

wherein b is an integer from 2 to 20, preferably from 3 to 10, most preferably 5.

The term "hydrocarbyl group" means an organic substituent consisting of carbon and hydrogen, which groups may include saturated, unsaturated or aromatic moieties.

Thus, preferred compounds of formula (I) are compounds of formula (Ia):

wherein X⁷is-NH₂Or

Wherein a is an integer from 1 to 10, preferably a is 1, and b is an integer from 2 to 20, preferably from 3 to 10, most preferably 5.

Preferred compounds of formula (II) are those wherein m is 0, n is 0, and Y is hydrogen.

The compounds of formulae (I) and (III) may be prepared by standard methods of peptide synthesis, for example solid phase peptide synthesis, for example as described by Atherton, e.g. and Sheppard, r.c.; "Solid Phase Synthesis"; IRL Press: oxford, 1989. Introduction of aminoxy groups into compounds of formula (I) can be achieved by preparation of a stable amide bond from the peptide amine functionality by reaction with an active acid and introduction during or after peptide synthesis.

In another aspect, the present invention provides compounds of formulae (I) and (Ia) as defined above, having use as agents useful in the production of radiolabelled peptidyl compounds.

In another aspect, the invention provides a compound of formula (III) or a salt thereof as defined above as a radiolabelled conjugate. Preferred compounds of formula (III) are compounds of formula (IIIa):

or a salt thereof, wherein X⁷is-NH₂Or

Wherein a is an integer from 1 to 10, preferably a is 1, and b is an integer from 2 to 20, preferably from 3 to 10, most preferably 5.

One particularly preferred compound of formula (III) is:

suitable salts of the compounds of formulae (III) and (IIIa) include pharmaceutically acceptable acid addition salts such as those formed from hydrochloric, hydrobromic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, oxalamic, fumaric, maleic, oxalamic, methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic and isoethanedioic acids.

The compounds of formula (II) may be prepared from the corresponding precursors of formula (IV) or protected derivatives thereof:

wherein L is a leaving group, preferably when m.gtoreq.1, L is p-toluenesulfonate, trifluoromethanesulfonate or methanesulfonate, or halide and when m is 0, L is p-trialkylammonium salt or p-nitro, and Y, m and n are as described for the compound of formula (II); preparation of aqueous [ alpha ], [ beta ] -an-olefin copolymer having a structure¹⁸F]Fluoride, in a suitable solvent such as acetonitrile, N-dimethylformamide or dimethyl sulfoxide, usually at ambient or elevated temperature, e.g. up to 140 ℃, by reaction with a base (e.g. from tetrabutylammonium or K₂CO₃Kryptofix-222) is suitably preactivated. The aldehyde or ketone functional groups of the compounds of formula (II) can also be generated rapidly from their protected precursors such as acetals or ketals by simple acid treatment after fluorination.

The compounds of formula (I) and (Ia) bind to receptors associated with angiogenesis as shown in the in vitro competitive binding assay below. These compounds may therefore be useful for in vivo diagnosis and imaging of angiogenesis-related therapies, diseases and disorders.

The term "angiogenesis-related diseases and conditions" includes those diseases and conditions referred to below. See also WO 98/47541 for this.

Diseases or disorders associated with angiogenesis include various cancers and metastases, such as breast, skin, colorectal, pancreatic, prostate, lung, or ovarian cancer.

Other diseases or conditions associated with angiogenesis are inflammation (e.g., chronic inflammation), atherosclerosis, rheumatoid arthritis, and gingivitis.

In addition, diseases or disorders associated with angiogenesis are arteriovenous reformations, astrocytomas, choriocarcinomas, glioblastomas, gliomas, hemangiomas (juvenile, capillary), liver cancers, endometrial hyperplasia, ischemic myocardium, endometriosis, kaposi's sarcoma, macular degeneration, melanoma, neuroblastoma, peripheral arterial occlusive disease, osteoarthritis, psoriasis, retinopathy (diabetic, proliferative), scleroderma, seminomas (semiomas), and ulcerative colitis.

The invention also provides a radiopharmaceutical composition comprising an effective amount (e.g. an effective amount for in vivo PET imaging) of a compound of formula (III) or (IIIa) as defined above or a salt thereof; and one or more pharmaceutically acceptable adjuvants, excipients or diluents.

A preferred embodiment of the invention relates to the use of a compound of general formula (III) or (IIIa) or a salt thereof as defined above for medicine, in particular for the in vivo diagnosis or imaging of diseases or disorders associated with angiogenesis by means of, for example, PET.

The radiolabeled conjugate of formula (III) or (IIIa) may be administered to a patient for PET imaging in an amount sufficient to generate the desired signal, typically a radionuclide dose of 0.01-100mCi, preferably 0.1-50mCi, most preferably 1-20mCi, will generally be sufficient for every 70kg body weight.

Thus, the radiolabeled conjugate of formula (III) or (IIIa) may be formulated for administration using a physiologically acceptable carrier or excipient in a manner well known to those skilled in the art. For example, the compounds, optionally with the addition of pharmaceutically acceptable excipients, may be suspended or dissolved in an aqueous medium and the resulting solution or suspension then sterilized.

Viewed from a further aspect the invention provides the use of a radiolabeled conjugate of formula (III) or (IIIa) or a salt thereof as defined above for the preparation of a radiopharmaceutical for use in an in vivo imaging method, suitable for PET, preferably for imaging of a disease or condition associated with angiogenesis; comprising administering said radiopharmaceutical to a human or animal body and generating an image of at least part of said body.

Viewed from a further aspect the invention provides an in vivo diagnosis or imaging for a disease or condition associated with angiogenesis comprising administering to said body, for example the vascular system, a radiopharmaceutical which has been distributed in the body using PET, and generating an image of at least a part of said body, wherein said radiopharmaceutical comprises a radiolabeled conjugate of formula (III) or (IIIa) or a salt thereof.

Viewed from a further aspect the invention provides a method of monitoring the effect of treatment of a human or animal body with a medicament, e.g. a cytotoxic drug, for a condition associated with cancer, preferably an angiogenic cancer. The method comprises administering a radiolabeled conjugate of formula (III) or (IIIa) or a salt thereof to the body and determining the uptake of the conjugate by cell receptors, preferably endothelial cell receptors and especially α ν β 3 receptors, the administration and determination being arbitrarily selected but preferably repeated, e.g. before, during and after treatment with the drug.

In a further embodiment of the invention there is provided a kit for the preparation of a radiofluoride tracer (radiofluorinated tracer) comprising a prosthetic group of formula (II) and a compound of formula (I).

In using the kit, the compound of formula (II) is added separately to the compound of formula (I), which may be suitably dissolved in an aqueous buffer (pH 1-11). After 1-70 minutes of reaction at a non-endpoint temperature, the labeled peptide can be purified and collected, for example, using Solid Phase Extraction (SPE) or High Performance Liquid Chromatography (HPLC).

Examples

The invention is illustrated by way of example, in which the following abbreviations are used:

HPLC: high performance liquid chromatography

NMR: nuclear magnetic resonance

TFA: trifluoroacetic acid

hr(s): hour(s)

min(s): minute (min)

DMAP: 4- (dimethylamino) pyridine

THF: tetrahydrofuran (THF)

DCM: methylene dichloride

DMF: n, N-dimethylformamide

TBAF: tetrabutylammonium fluoride

MeOH: methanol

TLC: thin layer chromatography

And (3) TIS: tri-isopropyl silane

DMSO, DMSO: dimethyl sulfoxide

PBS: phosphate buffered saline

PyAOP: [ 7-azabenzotriazol-1-yloxytris (pyrrolidinyl) hexafluorophosphate ]

Boc: tert-butyloxycarbonyl radical

RT: at room temperature

EXAMPLE 14 preparation of Trimethylammonium benzaldehyde triflate (Compound 1)

This compound was synthesized according to the method described by Haka et al (j. labelled Cpds. & radiopharmams 198927 (7) 823).

EXAMPLE 2 preparation of peptide precursor (Compound 2)

The peptide (compound 2) was synthesized using standard peptide synthesis methods.

(a)1, 17-diazide-3, 6, 9, 12, 15-pentaoxaheptadecane

A solution of dried hexapolyethylene glycol (25g, 88mmol) and methanesulfonyl chloride (22.3g, 195mmol) in dry THF (125mL) was cooled to 0 deg.C under an argon blanket in an ice/water bath. A solution of triethylamine (19.7g, 195mmol) in dry THF (25mL) was added dropwise over 45 minutes. After 1 hour the cooling bath was removed and the reaction was stirred for an additional 4 hours. Water (55mL) was then added to the mixture followed by sodium bicarbonate (5.3g to pH 8) and sodium azide (12.7g, 195 mmol). THF was removed by distillation and the aqueous solution was refluxed for 24 hours (two layers formed). The mixture was cooled, ether (100mL) was added and the aqueous phase was added sodium chloride toAnd (4) saturation. The organic phase was separated and the aqueous phase was extracted with ether (4X 50 mL). The organic phases were combined, washed with brine (2X 50mL) and dried (MgSO)₄). Filtration and evaporation of the solvent gave 26g (89%) of a yellow oil. The product was used in the next step without further purification.

(b) 17-azido-3, 6, 9, 12, 15-pentaoxaheptadecylamine

To a vigorously stirred solution of 1, 17-diazide-3, 6, 9, 12, 15-pentaoxaheptadecane (25g, 75mmol) in 5% HCl (200mL) was added a solution of triphenylphosphine (19.2g, 73mmol) in ether (150mL) over 3 hours at room temperature. The reaction mixture was stirred for an additional 24 hours. The phases were separated and the aqueous phase was extracted with dichloromethane (3X 40 mL). The aqueous phase was cooled in an ice/water bath and adjusted to pH 12 by the addition of solid potassium hydroxide. The aqueous phase was concentrated and the product was taken up in dichloromethane (150 mL). The organic phase was dried (Na)₂SO₄) And concentrated to give 22g (95%) of a yellow oil. The product was determined by electrospray mass spectrometry. (ESI-MS) (MH)⁺Calculated values: 307.19, respectively; found 307.4). The crude oil was used in the next step without further purification.

(c) 23-azido-5-oxa-6-aza-3, 9, 12, 15, 18, 21-hexaoxatriconic acid

To a solution of 17-azido-3, 6, 9, 12, 15-pentaoxaheptadecylamine (15g, 50mmol) in dichloromethane (100mL) was added diethanolic anhydride (Acros, 6.4g, 55 mmol). The reaction mixture was stirred overnight. The reaction was monitored by ESI-MS analysis and more reagent was added to drive the reaction to completion. The solution was concentrated to give a yellow residue, which was dissolved in water (250 mL). The product was separated from the aqueous phase by continuous extraction with dichloromethane overnight. Drying and evaporation of the solvent gave a yield of 18g (85%). The product was characterized by ESI-MS analysis. (MH)⁺Calculated values: 423.20, respectively; found 423.4). The product was used in the next step without further purification.

(d) 23-amino-5-oxa-6-aza-3, 9, 12, 15, 18, 21-hexaoxaicosaTriacid

23-azido-5-oxo-6-aza-3, 9, 12, 15, 18, 21-hexaoxatricosanic acid (9.0g, 21mmol) was dissolved in water (50mL) with H₂(g) -Pd/C (10%) reduction. The reaction was carried out until ESI-MS analysis showed complete conversion to the desired product (MH)⁺Calculated values: 397.2; found 397.6). The crude product was used in the next step without further purification.

(e) (Boc-aminoxy) acetyl-PEG (6) -glyoxylic acid

A solution of dicyclohexylcarbodiimide (515mg, 2.50mmol) in dioxane (2.5mL) was added dropwise to a solution of (Boc-aminoxy) acetic acid (477mg, 2.50mmol) and N-hydroxysuccinimide (287mg, 2.50mmol) in dioxane (2.5 mL). The reaction was stirred at room temperature for 1 hour and filtered. The filtrate was transferred to a reactor containing a solution of 23-amino-5-oxo-6-aza-3, 9, 12, 15, 18, 21-hexaoxatriconic acid (1.0g, 2.5mmol) and N-methylmorpholine (278. mu.l, 2.50mmol) in water (5 mL). The mixture was stirred at room temperature for 30 minutes. ESI-MS analysis showed complete conversion to the desired product (MH)⁺Calculated values: 570.28, respectively; found 570.6).

The crude product is purified by preparative HPLC (column: Phenomenex Luna 5. mu.C 18(2) 250X 21.20mm, detection: 214nm, gradient: 0-5% B within 60 minutes, where A ═ H₂O/0.1% TFA and B ═ acetonitrile/0.1% TFA, flow rates: 10 mL/min) to yield 500mg (38%) of pure product.

Detection by HPLC (column: Phenomenex Luna 3. mu.C 18(2) 50X 2.00mm, 214nm, gradient: 0-50% B in 10 min, where A ═ H₂O/0.1% TFA, and B ═ acetonitrile/0.1% TFA, flow rates: 0.75 mL/min, Rt 5.52 min) analyzed the product. Further confirmation was by NMR analysis.

(f) Conjugation of (Boc-aminoxy) acetyl-PEG (6) -glyoxylic acid with Compound 2

The reaction mixture of (Boc-aminoxy) acetyl-PEG (6) -glyoxylic acid (0.15mmol, 85mg) and PyAOP (0.13mmol,68mg) in DMF (2 mL). N-methylmorpholine (0.20mmol, 20. mu.L) was added and the mixture was stirred for 10 min. A solution of compound 2(0.100mmol, 126mg) and N-methylmorpholine (0.20mmol, 20. mu.L) in DMF (4mL) was added and the reaction mixture was stirred for 25 min. Additional N-methylmorpholine (0.20mmol, 20. mu.L) was added and the reaction mixture stirred for an additional 15 minutes. DMF is evaporated under vacuum and the product absorbs 10% acetonitrile-water and is checked by preparative HPLC (column: Phenomenex Luna 5. mu.C 18(2) 250X 21.20mm, UV214nm, gradient: 5-50% B in 40 min, where A ═ H₂O/0.1% TFA, and B ═ acetonitrile/0.1% TFA, flow rates: 10 mL/min) to yield 100mg (38%) of a semi-pure product. The second purification step, in which TFA was replaced by HCOOH (gradient: 0-30% B, otherwise identical to conditions described above), yielded 89mg (50%). HPLC (column: Phenomenex Luna 3. mu.C 18(2) 50X 2mm, detection: UV214nm, gradient: 0-30% B in 10 min, where A ═ H₂O/0.1% HCOOH, and B ═ acetonitrile/0.1% HCOOH, flow rates: 0.3 mL/min, room temperature: 10.21 minutes) the product was analyzed. The product was further characterized by ESI-MS (MH)⁺ calculated：905.4；found 906.0)。

Example 3- ¹⁸ Chemoselective combination of F-fluorobenzaldehyde with compound 3 to give compound 4

Deprotection of peptide 3 was performed by adding TFA containing 5% water to 10mg of peptide. Boc-deprotected peptide (5.9mg, 0.0044mmol) dissolved in 1mL of water was added to 4-fluorobenzaldehyde (compound 1) (1.1mg, 0.94. mu.l, 0.0089mmol) dissolved in 1mL of acetonitrile. The pH of the mixture was 3.5. After 45 min at 70 ℃ (degrees), the mixture is chromatographed using reverse phase preparative chromatography (phenomenex luna C18 column, 00G-4253-NO; solvent: a ═ water + 0.1% TFA/B ═ CH ═₃CN + 0.1% TFA, gradient: 30 minutesInner 10-40% B, flow rate 5.0 mL/min; detection at 214 nm) to yield 2.0mg (32%) of the pure compound (analytical HPLC: phenomenex Luna C18 column, 00G-4252-EO; solvent: a ═ water + 0.1% TFA/B ═ CH₃CN + 0.1% TFA, gradient: 10-50% B in 20 min, and the flow rate is 1.0 mL/min; retention time 16.3 min, detection at 214nm and 254 nm). Further characterization by mass spectrometry gave m/z value 1437.2. [ M-H ]⁺]。

Example 4: ¹⁸ radiosynthesis of F-Compound 4

The method comprises the following steps:

¹⁸f-fluoride (to 370MBq) in the presence of Kryptofix 222(5mg in 0.5mL acetonitrile) and potassium carbonate (50. mu.l, 0.1M in water) in N₂Heating to 110 ℃ under protection, and carrying out constant boiling drying for 20 minutes. At this point 3X0.5mL acetonitrile was added and evaporated to dryness. After cooling to < 40 ℃, trimethylammonium benzaldehyde trifluoromethanesulfonate solution (1mg in 0.4mL DMSO) was added. The reactor was sealed and heated to 90 ℃ for 15 minutes to effect labeling. At the same time, compound 3(6mg) was treated with 5% water in TFA (200. mu.l) for 5 min at room temperature. The solvent was then removed in vacuo. The deprotected peptide was redissolved in 0.1M NH₄OAc buffer, pH4(0.4mL), and 4-¹⁸F-fluorobenzaldehyde is mixed in a reactor. The reactor was sealed and heated to 70 ℃ for 15 minutes to effect coupling. After cooling to room temperature, the product was obtained by preparative radio-HPLC (column Phenomenex Luna C18(2)3 μm 10X 100mm, solvent: A ═ water/0.1% TFA and B ═ acetonitrile/0.1% TFA; gradient: 15-25% B in 5 min; 25% B12 min; 25-50% B in 10 min; flow rate 4.0 mL/min; UV detection at 210nm and 254 nm). The product fraction was diluted with water (10mL) and loaded onto a SepPakC18-plus cartridge (conditioned with 10mL EtOH and 20mL H2O). Compound 4 was eluted in ethanol (1 mL). Ethanol was removed in vacuo and compound 4 was formulated in PBS.

Method 2

a) ¹⁸ Radiosynthesis of F-fluorobenzaldehyde

¹⁸F-fluoride (up to 370MBq) in the presence of Kryptofix 222(5mg in 0.5mL acetonitrile) and potassium carbonate (50. mu.l, 0.1M aqueous solution) in N₂Heating to 110 ℃ under protection, and carrying out constant boiling drying for 20 minutes. At this point 3X0.5mL acetonitrile was added and evaporated to dryness. After cooling to < 40 ℃, trimethylammonium benzaldehyde trifluoromethanesulfonate solution (1mg in 0.4mL DMSO) was added. The reactor was sealed and heated to 90 ℃ for 15 minutes to effect labeling. The crude reaction mixture was cooled to room temperature and diluted with water. The mixture was passed successively through an ion exchange column (pretreated with ethanol (or acetonitrile) and water) eluting in an acetonitrile/water mixture. The eluate was concentrated using C18 Seppak and fluorobenzaldehyde was eluted in acetonitrile.

b) Compounds 3 and 4- ¹⁸ Conjugation of F-fluorobenzaldehyde

Compound 3 was treated with 5% trifluoroacetic acid in water for 5 minutes at room temperature. The solvent was then evaporated under vacuum. Peptide redissolving in 0.1M NH₄OAc buffer, pH4(0.5mL), and 4-¹⁸The F-fluorobenzaldehyde is combined in the reactor. The reactor was sealed and heated to 70 ℃ for 15 minutes to effect conjugation. After cooling to room temperature, the product was obtained by preparative radio-HPLC (as described in method 1) or by SPE.

Biological data

Binding study

Using cell membrane preparation methods known to express α v β 3 integrin receptors, and use¹²⁵I-echistatin and F-19 labeled peptide were used as competitive ligands for competitive binding studies. Binding curves were obtained and Kj values calculated using prism software.

Compound 4 had a Kj value of 10 nM.

Biodistribution of Lewis lung tumors

Lewis lung tumor (LLC) cells (0.1mL, 1X 107 cells/mL in culture) were injected subcutaneously into the right thigh of mice (male C57BL/6, approximately 25 g). Animals were controlled for tumor growth for up to 15 days, and this time was chosen for use in tumor model development because it now shows the highest concentration of angiogenesis.

To determine the biodistribution of the 18F-compound, the test drug (0.1mL, 5-10MBq/mL) was injected as an intravenous bolus into tumor-bearing animals via the tail vessels. Animals with two injections of different fold were treated with euthanasia. Dissect muscle, kidney, urine, lung, liver, stomach, small intestine, large intestine, thyroid, tumor, and extract blood samples. Dissected tissues and blood samples were weighed and counted (Wallac automated gamma particle counting system). At least three animals were studied at each time point. Results are expressed as% id and% id per gram of tissue.

Table 1 shows the biodistribution of compound 4 in a mouse Lewis lung tumor model. Summary data over time. The average data for 5 independent experiments are presented as mean (SD).

TABLE 1

Time (MINS P.I.)	Blood% ID/G	Muscle% ID/G	Lung% ID/G	Liver% ID/G	Tumor% ID/G
						5	6.35 (2.34)	1.76 (0.57)	6.54 (1.71)	6.01 (1.03)	2.69 (0.53)
60	0.84 (0.39)	0.56 (0.23)	2.12 (0.90)	1.48 (0.65)	1.84 (0.45)
						120	0.45 (0.13)	0.27 (0.07)	1.17 (0.28)	0.89 (0.29)	1.49 (0.32)

Time (MINS P.I.)	Tumor: blood, blood-enriching agent and method for producing the same	Tumor: muscle	Tumor: lung (lung)	Tumor: liver disease
					5	0.48	1.62	0.45	0.46
60	2.27	3.60	0.95	1.35
					120	3.31	5.80	1.27	1.75

As a control, table 2 shows the biodistribution of compound 5 in a mouse Lewis lung tumor model. Summary data over time. The average data for 5 independent experiments are presented as mean (SD).

TABLE 2

Time (MINS P.I.)	Blood% ID/G	Muscle% ID/G	Lung% ID/G	Liver% ID/G	Tumor% ID/G
						5	7.30 (1.3)	2.27 (0.6)	8.67 (1.4)	7.6 (0.9)	4.10 (0.9)
60	0.90 (0.2)	0.87 (0.3)	3.37 (0.5)	3.70 (0.9)	2.07 (0.3)
						120	0.71 (0.2)	0.44 (0.1)	2.03 (0.4)	3.28 (0.9)	1.12 (0.3)

Time (MINS P.I.)	Tumor: blood, blood-enriching agent and method for producing the same	Tumor: muscle	Tumor: lung (lung)	Tumor: liver disease
					5	0.6	1.8	0.5	0.5
60	2.3	2.6	0.6	0.6
					120	1.6	2.6	0.6	0.3

The additional PEG moiety of compound 4 imparts significantly more favorable in vivo properties. In particular, the residual activity of compound 4, present in background tissues such as blood, muscle, lung and liver, was substantially less than the activity of compound 5 after 120 minutes. Subsequent tumors were: the background ratio is significantly improved and thus imaging is enabled.

Claims (16)

1. A method of radiofluorination comprising reacting a compound of formula (Ia):

wherein X⁷is-NH₂Or

Wherein a is an integer of 1 to 10 and b is an integer of 2 to 20.

Reaction with a compound of formula (II):

wherein:

n is an integer of 0 to 20;

m is an integer of 0 to 10;

y is hydrogen, C_1-6Alkyl, or phenyl

To give a compound of formula (IIIa):

wherein m, n and Y are as defined for the compound of formula (II), X⁷And b is as defined for compounds of formula (Ia).

2. The method according to claim 1, wherein a is 1.

3. The method according to claim 1, wherein b is 3 to 10.

4. The method according to claim 1, wherein b is 5.

5. A compound of formula (Ia) as defined in claim 1.

6. A compound of formula (IIIa):

or a salt thereof, wherein X⁷is-NH₂Or

Wherein a is an integer of 1 to 10 and b is an integer of 2 to 20.

7. The compound according to claim 6, wherein a is 1.

8. The compound according to claim 6, wherein b is 3-10.

9. The compound according to claim 6, wherein b is 5.

10. The compound according to claim 6, which is:

11. a radiopharmaceutical composition which comprises an effective amount of a compound of formula (IIIa) or a salt thereof as defined in any one of claims 6 to 10 together with one or more pharmaceutically acceptable adjuvants, excipients or diluents.

12. Use of a radiolabeled conjugate of a compound of formula (IIIa) or a salt thereof as defined in any one of claims 6 to 10 in the manufacture of a radiopharmaceutical suitable for PET for use in a method of in vivo imaging, wherein said radiopharmaceutical is administered to the human or animal body and produces at least part of said body image.

13. The use as defined in claim 12, for imaging a disease or condition associated with angiogenesis.

14. Use of a compound of formula (IIIa) or a salt thereof as defined in any one of claims 6 to 10 in the manufacture of a radiopharmaceutical for in vivo diagnosis or imaging of a disease or condition associated with angiogenesis.

15. Use of a compound of formula (IIIa) or a salt thereof as defined in any one of claims 6 to 10 in the manufacture of a radiopharmaceutical for monitoring the therapeutic effect of the drug against a cancer-related condition in the human or animal body.

16. The use as defined in claim 15, wherein said medicament is against angiogenesis.